The Role Of Epigenetic Regulation Of Bdnf Gene Transcription In Radiation-induced Neurogenesis Damage And Cognitive Dysfunction

Part I. Whole Brain Irradiation Induced Acute Cognitive Deficit andHistone H3Acetylation Decline in RatPurpose/Objective(s): Radiotherapy is one of the predominant methods for thetreatment of primary and secondary brain tumors. Unfortunately, radioactive neurotoxicityin normal brain affects the quality of life (QOL) for cancer survivors. Acute and chroniccognitive decline have been observed in patients who survive post-radiotherapy. Acutecognitive dysfunction has become the barriers to improve therapies and prophylaxes forhead and neck cancer. The present study aimed to investigate the effect of single dose of2Gy,10Gy, and30Gy whole brain irradiation (WBI) on acute cognitive function inSprague-Dawley rats and to investigate the role of epigenetics in the irradiation-inducedcognitive deficiency by observing the alterations of histone acetylation in the hippocampusof rats after irradiation.Materials/methods: A signal dose of2Gy,10Gy and30Gy4MV electron beam weregiven to adult male Sprague-Dawley rats (150–200g), this dose covered a dose range ofclinical and experimental applications, and the animals were divided into2Gy group,10Gygroup,30Gy group, and control group. To evaluate the consequence of whole brainirradiation on hippocampus-dependent memory formation, Morris water maze test,passive-avoidance test and open field test were performed. After whole brain irradiation,the total protein was extracted from irradiated hippocampus and whole brain30μmcryosections were cut by using a cryostat at day7and day30. Western blot was performedand the brain sections were immunohistochemical stained. The alterations of histone H3acetylation was observed and analyzed.Results: Our date found2Gy and10Gy WBI did not induce any cognitive deficits. Results showed that there was no significant difference between irradiated andnon-irradiated groups in performance of open field test (P>0.05). However,30Gy led tosignificant deficits of spatial memory, passive avoidance behavior and retention memory ofrats by Morris water maze test, passive-avoidance test one month postirradiation.Consistent with the neurological changes,30%(P <0.05) and61%(P <0.01) reduction ofhistone H3acetylation was observed at7and30days after30Gy radiation in atime-dependent manner. Interestingly, we found that irradiation did not impair theexpression of HDAC2, which has been indicated negatively regulates memory formationand synaptic plasticity. However, HDAC1increased as early as7thday and reached higherlevel one month postradiation.Conclusion: In summary, different from previous studies, this study found30Gy WBI,but not2and10Gy, induced significant cognitive deficit and HDAC1-dependent H3acetylation decline in hippocampus. These findings suggest that a single-dose exposure at30Gy is sufficient to induce acute cognitive dysfunction in rat, and this injury may beassociated with alterations in histone H3acetylation in hippocampus. Their role in thepathogenesis of radiation-induced injury needs to be further explored.Part II. Irradiation-induced Hippocampal Neurogenesis Impairment IsAssociated with Epigenetic Regulation of bdnf Gene TranscriptionObjective(s): Radiation-induced cognitive decline affects the quality of life for cancersurvivors. There are evidences that neurogenesis impairment in hippocampus play a keyrole. But the pathogenesis remains unknown. This study aimed to detect potentialpathogenesis of radiation-induced neurogenesis impairment by investigating whetherradiation inhibits BDNF-Trkb signaling in hippocampus, and to reveal whether or not bdnfgene transcription was repressed via histone acetylation.Methods: Our previous study (Part I) demonstrated30Gy whole brain irradiation(WBI) induced significant cognitive deficit in SD rat, but not2and10Gy. So, in the study,the irradiation brain injury model was used. A single dose of30Gy4MV electron beamwas given to male SD rats. After7and28days, immunohistochemistry was used toanalyze the proliferation of neural precursor cells and long-term survival of the new bornneurons. Proliferating neurons were labeled with5-bromo-2Vdeoxyuridine (BrdU) and NeuN. Confocal microscopy was used to determine the percentage of BrdU-labeled cellsthat showed mature cell phenotypes. AT the same time, the total protein was extracted fromradiated hippocampus, and western blot was performed to analyze the expression of BDNFand Trkb. Transcription of bdnf gene is controlled by a series of promoters regulated byepigenetic modifications. We next performed ChIP assay with RT-PCR to measureacetylated histone at the promoter region of bdnf gene. To determine whether WBI has aneffect on the CpG methylation pattern within the bdnf gene, we assessed the methylationstatus within55942bp. The region covers the promoter, first and second exon, which hasbeen shown to be crucial for bdnf expression regulation. We performedbisulfite-sequencing analysis in these CpG sites. Finally, TSA (0.1mg/kg), a HDACsinhibitor, was used with once daily with subcutaneous injections of trichostain, then weverified whether or not bdnf transcription and neurogenesis impairment was rescued.Results: Immunofluorescence showed WBI seriously damaged the production of newneurons and long-term survival, which correlated with decreased mRNA and proteinexpression of bdnf. At the7thday after radiation, compare to control, BrdU and NeuNdouble positive cells in radiated rats reduced67%(P <0.01), at the28thday, there arealmost no BrdU and NeuN double positive cells were observed. Western blot analysisshowed BDNF and Trkb in hippocampus decreased significantly after radiation. We founda33%reduction of BDNF at the7thday (P <0.05) and51%reduction at the30thday (P <0.05). Consistently, as specific receptor of BDNF, Trkb respectively reduced24%(P <0.05) and35%(P <0.05). Consistently, a significant decrease of H3acetylation wasobserved at bdnf promoters of exon I, II and V in ChIP analysis. TSA, a HDACs inhibitor,triggered bdnf transcription and rescued neurogenesis impairment. No significant alterationof methylation was detected in the irradiated animals compared to control.Conclusion: In summary, the data firstly provide evidence radiation inducedpersistent inhibition of BDNF-TrkB signaling through HDAC1-dependent H3acetylationdecline, which is associated with long-term impairment of neurogenesis in DG. The resultalso raises a possibility that triggering BDNF-TrkB signaling by inhibiting HDAC-1maybe used to stimulate neurogenesis. This will be help to devise new therapies againstradiation-related cognitive deficits.